Effects of Epidermal Growth Factor on Cytosolic Calcium Levels and Phosphoinositide Metabolism in Normal Rat Osteoblastic Cells. Epidermal growth factor (EGF) is a bone resorptive agonist known to be involved in the regulation of intracellular calcium via inositol (1,4,5) triphosphate (IP3) production in some cell types. Whether a similar mechanism of action of EGF occurs in normal osteoblastic cells remains to be elucidated. The aim of this study was to evaluate the effects of EGF on cytosolic calcuim levels in normal rat osteoblastic cells and to study the possible involvement of changes in phosphoinositide metabolism in the regulation of this ion. Cells were obtained from rat calvaria by sequential collagenasedigestions and grown in BGJb media until confluency. Cytosolic calcuim measurements were done in fura-2 loaded cells using a SPEX Fluorolog II spectrofluorometer. The phosphoinositide analyses were performed on cells labeled with (3H)-inositol, and HPLC analyses were conducted. Radioactive counts corresponding to the IP, IP2, IP3 and IP4 peaks were added together and the individual peaks obtained were expressed as a percentage of inositol phosphate counts. Data were analyzed using ANOVA and Student's t-test. EGF (10nM) elicited significant increases in cytosolic calcium levels. There appears to exist a rapid, transient increase (dependent on extracellular calcium) as well as a slower, sustained component to the EGF effect. The sustained component was inhibited by the phospholipase C inhibitor, U73122. The addition of EGF (50nM) to the cells produced significant (p<0.005) changes in phospho-inositide levels compared to the controls: the percentage of IP2 counts was increased at 20, 45, 180 and 300 secs. However, the percentage of IP3 counts, was not significantly increased, even though a tendency towards an increase was noticed at 90 sec. This increase was coincident with that of the slower, PLC-dependent component of the EGF-calcium effect. These results suggest that EGF's mechanism of action in normal osteoblastic cells in increasing cytosolic calcium levels is due, only in part, to increases in phosphoinositide metabolism. A substantial component of the calcium response appears to be regulated by an IP3-independent mechanism. Key Words: EGF-Epidermal Growth Factor; inositol triphosphate; IP3; osteoblastic cells; phosphoinositide; phospholipase C; cytosolic calcium